以電泳膠體分離及質譜儀鑑定紅斑性狼瘡(活躍期)血漿中表現差異的蛋白質

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李怡萱(Yi-Syuan Li) 查詢紙本館藏

畢業系所

系統生物與生物資訊研究所

論文名稱

以電泳膠體分離及質譜儀鑑定紅斑性狼瘡(活躍期)血漿中表現差異的蛋白質
(Identification of proteins with different expression in plasma from active systemic lupus erythematosus by gel electrophoresis and mass spectrometry)

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摘要(中)

紅斑性狼瘡是一種具有多種臨床病症，由自體免疫異常所導致的自體免疫性疾病，
屬於風濕病的其中一種。紅斑性狼瘡症狀包含皮疹、發燒、關節炎、感到疲倦及腎絲球
炎及神經症狀等，紅斑性狼瘡症狀與其他疾病相似，因此容易誤診。本實驗主要目的為
尋找台灣紅斑性狼瘡與健康人體中不同的蛋白質，可提供具潛力的生物標記分子，應用
於此疾病之診斷上。國泰綜合醫院過敏免疫科提供血漿樣本，共有19 位病人 (包含18
位女性與1 位男性) 及12 位健康對照組 (包含9 位女性與3 位男性)，病人平均年齡為
32.1±11.5，健康對照組之平均年齡為41.9±12.2。利用蛋白質體學的方法，固定血漿蛋
白總量，並以1 維與2 維膠體電泳法將病患及健康對照組的蛋白質進行分離，以影像分
析將蛋白質表現量進行統計分析，病人與健康對照組的數值比較，選取具差異的蛋白質
(U-test, p<0.5)，從膠體上切下，經過蛋白質水解酵素水解後，利用兩種質譜儀 (MALDI
Q-TOF and ESI-MS/MS) 進行蛋白質體身份鑑定。此實驗中，一共找到15 個蛋白質，9
個為表現量增加的蛋白質。其中9 個蛋白質是文獻已發表和紅斑性狼瘡有關，顯示本方
法可快速全面檢視表現差異蛋白質。而6 個蛋白質為本研究首次發現與紅斑性狼瘡有關，
顯示本方法可提供其他方法無法測得之差異蛋白質。本研究提供了快速尋找新紅斑性狼
瘡具潛力的生物標記分子的方法，將來也可能可以應用於利用血漿樣品尋找其他疾病的
潛力蛋白質標記分子。

摘要(英)

Systemic lupus erythematous (SLE) exhibits an aggressive clinical phenotype with
severe complications and overall poor prognosis. Common initial and chronic complaints
include fever, joint pains, fatigue and temporary loss of cognitive abilities. These symptoms
are so similar with other disease, so it is very difficult to diagnose and treat. The goal of this
study is to identify significant different proteins between SLE patients compared to healthy
people, the significant proteins may help to diagnose SLE patients. The SLE plasma samples
which included male and 18 female patients, with average age 32.1±11.5 also diagnosed with
renal involvement. The SLE plasma samples were collected from Cathay General Hospital,
Taipei, Taiwan. The protein profile from patients was randomly compared to that from twelve
healthy controls from 3 male and 9 female with average age at 41.9±12.2. The protein
concentration in the plasma samples was quantified and the total protein was separated by
one-dimensional and two-dimensional SDS-polyacrylamide gel electrophoresis. The up- and
down-regulated proteins compared to healthy plasma (U-test, p < 0.5) were selected and cut
from the gels and digested with trypsin for enzymatic analysis of protein using mass
spectrophotometry for identification of proteomic analysis followed by the amino acid
sequencing with mass spectrometry (MALDI-TOF/TOF and ESI-MS/MS). This experiments
results a total of 15 proteins found, 9 proteins are up-regulated and 6 proteins are down
regulated. In total of 15 proteins, nine proteins are published relation with lupus and six
proteins are new.These new proteins in the SLE patients may contribute to find the new
potential lupus biomarkers determine the precise role of newly identified SLE-related proteins
pathogenesis and their function as biomarkers will require further study.

關鍵字(中)

★ 紅斑性狼瘡
★ 蛋白質體學
★ 膠體電泳

關鍵字(英)

★ systemic lupus erythematosus
★ proteomic
★ electrophoresis

論文目次

中文摘要 I
Abstract III
I: Introduction: 1
1.1 Autoimmune disease 1
1.2 Systemic lupus erythematosus 2
1.3 Diagnosis 3
1.4 Treatment 5
1.5 Prognosis 6
1.6 Biomarkers 7
1.7 SLE biomarkers 8
1.8 Research aim: 12
II: Materials and methods 14
2.1 Preparation of proteins from samples 14
2.1.1 Protein dilution 14
2.1.2 Proteins quantitation 15
2.2 One-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (1D SDS-PAGE) 16
2.3 SDS-PAGE image analysis and statistics 17
2.4 Two-dimensional gel electrophoresis 17
2.5 2-DE gel image analysis and statistics 18
2.6 In-gel digestion 19
2.7 Mass spectrometry 19
2.7.1 MALDI-TOF 20
2.7.2 LC ESI MS/MS 20
2.8 Database searching 21
III: Result 24
3.1 The laboratory main parameters of 19 SLE patients and 12 healthy controls 24
3.2 Variation in plasma protein expression between lupus patients and healthy controls in 1D SDS-PAGE 24
3.3 Identification of the proteins in 1D SDS-PAGE by MALDI TOF/TOF mass and LC-ESI MS/MS 24
3.4 Variation in plasma protein expression between lupus patients and healthy control in 2D electrophoresis 25
3.5 Identification of the protein in 2D electrophoresis by MALDI TOF/TOF mass 27
IV: Discussion 27
V: Conclusion 32
VI: Reference 34
VII: Figure 41
41
VII: Table 104
VIII: Appendix 108

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指導教授

黃雪莉(Shir-Ly Huang)

審核日期

2013-8-28

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